Fig 1: The antiviral effect of MTX is a result of defective purine synthesis, which can be rescued by folinic acid or a purine nucleoside (A) Metabolic map indicating the major steps inhibited by MTX. Enzymes are indicated in italics. Enzymes directly inhibited by MTX are printed in bold and italics. Plain arrows indicate direct reactions, dashed arrows indicate reactions with intermediates that are omitted for simplicity. Metabolites used for rescue experiments are shown with a gray background. Folic acid metabolites: DHF, dihydrofolate; THF, tetrahydrofolate. Folic acid metabolism, enzymes:DHFR, dihydrofolate reductases; MTHFR, methylene tetrahydrofolate reductase; TYMS, thymidylate synthetase. Purine synthesis, enzymes:GARFT, glycinamide ribonucleotide formyltransferase; AICARFT, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase, part of the dual function enzyme ATIC (comprising AICARFT and IMP cyclohydrolase). Purine synthesis, metabolites: PRPP, phosphoribosyl pyrophosphate; GAR, glycinamide ribonucleotide; FGAR, formylglycinamide ribonucleotide; AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; FAICAR, 5-formamidoimidazole-4-carboxamide ribonucleotide. Nucleotides are indicated by standard abbreviations, i.e. IMP (inosine monophosphate), AMP, GMP, ATP, GTP, dUMP, dTMP. Amino acids are abbreviated as Ser, Gly, Met. (B) ATP levels upon MTX-treatment and SARS-CoV-2 infection. Vero cells were treated with MTX as indicated and infected with SARS-CoV-2 as in Fig. 2. Cells were counted using the Celígo® S Imaging cytometer and ATP levels were measured by the CellTiter-Glo® assay. This experiment was performed on four separate plates with four technical replicates each (mean with SD). The extent of luminescence, reflecting relative ATP levels, was normalized to the number of cells found under the same conditions and further normalized to untreated and mock-infected cells. Thus, the columns reflect the relative amount of ATP per cell in each case. Note that SARS-CoV-2 infection as such did not significantly affect ATP levels. (C) Rescue of virus replication in the presence of MTX by folinic acid (leucovorin). Vero cells were inoculated with virus as in Fig. 2, with or without MTX treatment 24 hrs before and during infection. In parallel or in addition, leucovorin was added to the cell culture media, at a concentration of 10 µM (mean with SD, n = 3). (D) Restored virus replication by inosine, in the presence of MTX. The experiment was carried out as in (A), with the addition of 1 mM inosine instead of leucovorin (mean with SD, n = 3). (E) Rescue of virus protein synthesis by leucovorin or inosine, in the presence of MTX. Upon treatment and infection of Vero cell as in (A) and (B), cell lysates were subjected to immunoblot analysis. The viral S and N proteins, DHFR as well as GAPDH and Hsc70 (loading controls) were detected.
Fig 2: Excluding cell cycle arrest as the major antiviral mechanism of MTX (A) Lack of measurable cytotoxicity by MTX. Vero cells were treated with MTX at the indicated concentration for 72 hrs. The release of lactate dehydrogenase (LDH) to the supernatant was quantified by bioluminescence as a read-out for cytotoxicity. The percentages reflect the proportion of LDH released to the media, compared to the overall amount of LDH in the cells. The number of cells per well were quantified by the Celígo® S Imaging cytometer. Note that the number of cells was reduced after treatment with higher concentrations of MTX but cytotoxicity levels remained unchanged (mean with SD, n = 3). Thus, the CC50 (concentration to kill 50% of all cells) was never reached in these experiments, even with the highest concentration of MTX, i.e. 10 µM. The reported CC50 for MTX is >90 µM (Beck et al., 2019; Fischer et al., 2013). (B) Increased levels of dihydrofolate reductease (DHFR), p53 and p21 upon MTX treatment, regardless of virus infection, accompanied by decreased virus protein synthesis in the presence of MTX. Upon MTX or palbociclib treatment and/or infection of Vero cells as in Fig. 2, the viral N and S proteins as well as DHFR, p53, p21, CDK4 and GAPDH (loading control) were detected by immunoblot analysis. Note that the virus protein levels remained unchanged upon palbociclib treatment. (C) Induction of a cell cycle arrest upon MTX and palbociclib treatment. Vero cells were treated with 1 µM MTX or palbociclib and infected with SARS-CoV-2 as in Fig. 2. Flow cytometry was performed to analyze cell cycle progression. Note that both drugs induced a cell cycle arrest and therefore impaired cell proliferation. (D) Reduction of virus RNA progeny by MTX but not palbociclib. Vero cells were treated with 1 µM MTX/palbociclib, infected as in Fig. 2. and the virus RNA was detected by quantitative RT-PCR. MTX was found capable of reducing virus RNA yield by more than 1000-fold, whereas palbociclib did not diminish virus replication (mean with SD, n = 3).
Fig 3: Western blotting and immunohistochemistry assay of cortex and striatum regions to quantify GTPCH and DHFR expressions. A Immunoblotting of GTPCH and DHFR in the cerebral cortex and striatum tissues of R6/2 and NCAR mice using ?-tubulin as internal control (n = 4). Protein bands were quantified as described in Fig. 4. Original blots before cropping are presented in Fig. S12. B Diagram of mouse brain section divided into five regions: three regions of cortex (I-III); striatum (IV); hypothalamus and pallidum (V). C, D GPTCH and E, F DHFR positive cells and their ratios were quantified in five regions (I–V). *p < 0.05; **p < 0.01
Fig 4: C1 metabolic pathway and characterization of R6/2 mice. A Folate, Met and BH4 cycles in plants and animals and their associated metabolism. Red lines stand for mammal specific, green lines stand for plant specific while black lines stand for both. All enzymes with protein levels examined by immunoblotting are marked in red. B mHtt protein aggregates in cortex and striatum regions were detected with anti-Htt antibody (mEM48) in 4-week-old male R6/2 and NCAR mice. C, D Quantification analysis of immunoblotting results of GTPCH, DHFR, QDPR, MS, MAT1/2A, AHCY, MTHFR, TPH2, TH, nNOS and ChAT (n = 7). The band intensity of each protein from western blotting D was normalized with ?-tubulin on the same blot. The ratio was further calculated against NCAR whose relative expression level was set as 1. All data plotted are the average (n = 7) ± SD. Only one representative western blotting of ?-tubulin is shown. Original blots of above proteins before cropping are presented in Fig. S11. E Contents of BH4 and BH2, and their ratio in brain tissues and plasma (n = 4, average ± SD). *p < 0.05; **p < 0.01. ***p < 0.001. Abbreviations used for enzymes: AHCY S-adenosylhomocysteine hydrolase, ChAT choline acetyltransferase, DHFR dihydrofolate reductase, GTPCH GTP cyclohydrolase I, MAT1/2A methionine adenosyltransferase, MS methionine synthase, MTHFR methylene-tetrahydrofolate reductase, nNOS neuronal nitric oxide synthase, QDPR quinoid dihydropteridine reductase, TH tyrosine hydroxylase (Tyr), TPH2 tryptophan hydroxylase
Fig 5: In vitro, inhibition of NOX2 in astrocytes after SAH recouples eNOS by increasing the expression of DHFR in endothelial cells. In vitro, LV-shDHFR largely attenuated the protective effect of NOX2 inhibitors after SAH. (A) NO released by cells among different experimental groups. Data are shown as mean ± SD (n = 6 each group, ***p < 0.001, ns: no significant difference). (B,C) Representative Western blot images and quantitative analysis of eNOS monomer and eNOS dimer expression by ImageJ. Data are shown as mean ± SD (n = 6 each group, ***p < 0.001, ns: no statistically significant difference). (D,E) Representative Western blot images and quantitative analysis of endothelial DHFR expression by ImageJ. Data are shown as mean ± SD (n = 6 each group, **p < 0.01, ns: no statistically significant difference). (F) Immunofluorescence images showed CD31 and DHFR colocalization. All scale bars, 50µm. (G,H) Representative Western blot images and quantitative analysis of endothelial DHFR expression by ImageJ. Data are shown as mean ± SD (n = 6 each group, ***p < 0.001). (I,J) Representative Western blot images and quantitative analysis of eNOS monomer and eNOS dimer expression by ImageJ. Data are shown as mean ± SD (n = 6 each group, ***p < 0.001). (K) NO released by cells among different experimental groups. Data are shown as mean ± SD (n = 6 each group, ***p < 0.001, ns: no statistically significant difference).
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